The CFTR is a transmembrane protein found in the epithelial cells of mucosal surfaces. It is involved in the transport of chloride and bicarbonate ions across epithelial membranes where it functions as an ATP-gated ion channel and thereby also influences the balance of other ions such as sodium across such membranes. When an appropriate ionic balance is maintained, the mucus layer of the mucosal surface displays a normal structure and composition and therefore behaves and functions normally. Insufficient numbers of functional CFTR ion channels at the epithelial cell surface, e.g. as a consequence of mechanisms which cause reduced numbers of CFTR at the cell surface, and/or insufficient ion channel activity in the population of CFTR that are present at the cell surface, results in the pathological state referred to as CFTR (ion channel) dysfunction. The perturbations in ionic balance at mucosal surfaces caused by CFTR dysfunction manifest in stagnant mucus in all organs where mucus is formed, and thickened secretions from glands in the liver and the pancreas. The presence of this stagnant mucus in the lungs, paranasal sinuses, gastrointestinal (GI) tract, pancreas, liver and female and male reproductive systems leads to a plethora of clinical conditions associated not only with poor quality of life but also morbidity and mortality. Indeed, in cystic fibrosis, the most recognisable disease associated with CFTR dysfunction, sufferers typically succumb to a complication directly associated with this stagnant mucus.
CFTR dysfunction, or more specifically CFTR ion channel dysfunction (which terms are considered synonymous for the purposes of the invention and are therefore used interchangeably herein) typically arises from a defect in the CFTR which affects its activity and/or its cellular processing and delivery (trafficking) to the cell surface. Such defects may in many cases be due to mutations in the CFTR (i.e due to an underlying genetic defect), but can also arise due to extrinsic factors which may for example cause aberrant or impaired expression of the CFTR at the cell surface. Taking these various “defects” together, it can be seen that, broadly speaking, six classes of “mechanism” underlying defective CFTR function can be recognised, and CFTR dysfunction may arise from one or more of these. Class I mechanisms involve the presence of a premature stop codon in the CFTR mRNA transcript and give rise to truncated CFTR with reduced function and/or which are poorly transported to the cell membrane. Class II mechanisms involve impaired intracellular processing of full length CFTR translation products which interferes with the CFTR's route to the cell membrane (e.g. misfolding, defective post-translational modification, inappropriate intracellular protein sorting, degradation prior to reaching the cell membrane). Class III mechanisms involve disordered ion channel regulation (e.g. poor activation by ATP or cAMP, reduced channel open time). Class IV mechanisms involve reduced channel conductance. Class V mechanisms involve splicing defects in the transcription of CFTR mRNA or reduced CFTR mRNA transcription per se. Like Class I, Class V mechanisms can give rise to CFTR with reduced function and/or reduced quantities of CFTR in the cell membrane. Class VI mechanisms involve accelerated turnover of CFTR protein at the cell membrane which reduces the quantities of CFTR in the cell membrane.
CF is an autosomal recessive genetic disease of humans arising from mutations in the CFTR which result in CFTR dysfunction. CF-causing mutations may be classified within different “mechanistic” classes as set out above and thus the aetiology of CF is extremely diverse.
In the lungs of CF patients, the dense, attached and intractable mucus caused by the CFTR dysfunction is insufficiently cleared by the mucociliary clearance system, and accumulates in the airways. This makes patients susceptible to chronic lung infections and inflammation (pneumonia), which causes bacteria, bacterial biofilm, and cell debris to become intermixed with the mucus and leads to increased mucus viscosity. In turn this eventually leads to permanent lung damage and remodelling and further to pulmonary hypertension, heart failure, and respiratory failure. Infection by Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, Mycobacterium avium complex and Aspergillus fumigatus are common. Abnormal mucus higher up in the respiratory tract (e.g. in the bronchi) can also be susceptible to infection which in turn may lead to inflammation of mucosal surfaces (e.g. bronchitis). Response to antibiotics is often poor.
In the paranasal sinuses the abnormal mucus results in frequent blockages leading to facial pain, headaches and abnormal nasal drainage. The sinuses are often exacerbated by infection, to which the abnormal mucus is highly susceptible and this may lead to acute, subacute and chronic sinusitis (also known as rhinosinusitis). Overgrowth of the nasal tissue (nasal polyps) may also result as a consequence of the chronic inflammatory state induced from chronic sinus infection. These polyps can block the nasal passages and increase breathing difficulties.
In the GI tract the attached and abnormal mucus is thought to result in intestinal pain and even full obstruction. In neonatal subjects mucus can combine with meconium to plug the ileum (meconium ileus). In older patients intestinal blockage by intussusception and distal intestinal obstruction syndrome (DIOS) of the distal ileum is often seen. Bacterial overgrowth and complications associated with the stagnant mucus may also occur.
In the pancreas, thickened and attached mucus in exocrine secretions often blocks the pancreatic duct and reduces the amount of digestive enzymes and bile entering the GI tract. This causes accumulation of digestive enzymes in the pancreas which in turn reduces the ability of a patient to retrieve dietary nutrients (nutrient malabsorption) and can cause inflammation, and irreversible damage to the pancreas. Such inflammation and damage results in pancreatitis (both acute and chronic) and ultimately atrophy of the exocrine glands and fibrosis. Damage of the pancreas can also lead to loss of the islet cells, leading to cystic fibrosis-related diabetes.
In the liver thickened bile secretions and mucus lining may block the bile ducts, causing gallstones, and lead to liver damage and ultimately cirrhosis.
Fertility of healthy females is regulated in part by the properties of the mucus in the reproductive system, especially the mucus of the cervix. The vas deferens of male mammals contains mucus that can obstruct the vas deferens if that mucus is abnormal. The abnormal mucus caused by mutation in the CFTR gene has therefore been connected with both female and male infertility.
There is currently no cure for the underlying genetic causes of CF, although the life-threatening lung and liver disease can sometimes be resolved with a successful lung or liver transplant. Lung transplants in CF patients are however not always successful because lung infection can recur shortly after transplantation. This is usually a consequence of the use of immunosuppressant drugs to promote establishment of the transplant making the transplant susceptible to the infections that remain in the patient's respiratory tract above the newly transplanted lungs.
Other conditions beyond CF may also be characterised by, or associated with, CFTR dysfunction. In recent years it has been recognised that even in subjects who do not suffer from classical or “overt” CF (that is who do not carry homozygous or compound heterozygous mutations in their CFTR alleles) CFTR dysfunction at epithelial cell layers can occur and give rise to the abnormal mucus and endocrine secretions that are like or similar to those that characterise CF. Thus, for example such subjects may carry one or more CFTR mutations, but the mutations do not result in CFTR dysfunction of sufficient severity to qualify as CF as such. This results in abnormal mucus clearance which in turn may lead to or at least contribute to, inter alia, breathing difficulties, CF-like symptoms and complications, and chronic inflammatory respiratory disorders including COPD (and its subtypes chronic bronchitis and emphysema), bronchiectasis, asthma and chronic sinusitis. Accordingly, diseases and disorders (or more generally a “condition”) associated with CFTR dysfunction may include not only CF, but also other conditions involving respiratory dysfunction (more generally other respiratory disorders), and in particularly disorders involving pulmonary obstruction, including particularly asthma.
In some instances CFTR dysfunction is seen in subjects that have non-compound heterozygous mutant CFTR alleles. In such subjects the inherited dysfunction is mild and so is insufficient to manifest as overt CF, but is sufficient to result in mucus that is more dense, attached and intractable than normal, as well as secretions from glands in the liver and the pancreas that are thicker than normal. As discussed above in the context of overt CF, in the respiratory tract, such mucus is often insufficiently cleared by the mucociliary clearance system and so accumulates in the airways and may lead to further symptoms and complications. Similarly, the thickened mucus and exocrine secretions in the paranasal sinuses, gastrointestinal (GI) tract, pancreas, liver and female and male reproductive systems of these subjects may be sufficient to lead to mild forms of the plethora of clinical conditions associated with overt CF.
In other instances it has been shown that CFTR dysfunction may be acquired. It is now known that the chronic inhalation of particulate irritants, e.g. smoke particles (tobacco, wood etc.), pollution, dust (asbestos, cotton, coal, stone, animal droppings etc.) and spores, can result in reduced CFTR ion channel activity (e.g. reduction in gating and/or conductance) at epithelial cell surfaces carrying the receptor. It will be seen that in subjects who display mild CFTR dysfunction because of an inherited defect, these deleterious effects of environmental factors on CFTR may be more pronounced clinically. This acquired dysfunction and the effects on mucus are thought to contribute to the progression of chronic inflammatory disorders, e.g. COPD, CB, emphysema, bronchiectasis and chronic sinusitis in these subjects. It has also been recognised that the intracellular processing of CFTR can be interfered with and the turnover of CFTR at the cell membrane can be accelerated during chronic airway inflammation, e.g. as seen in COPD (and its subtypes chronic bronchitis and emphysema), bronchiectasis and chronic sinusitis.
In all of these contexts CFTR dysfunction in the respiratory tract may result in the dense, attached and intractable mucus characteristic of CF which is insufficiently cleared by the mucociliary clearance system and which accumulates in the airways. This makes patients with acquired CFTR dysfunction susceptible to the respiratory symptoms and complications experienced by CF patients, including those shared with COPD, CB, emphysema, asthma and chronic sinusitis.
Until recently, pharmaceutical intervention in CF and other conditions associated with CFTR dysfunction has been restricted to management of secondary symptoms and conditions and very few options are available to address the main underlying cause of those conditions: the abnormal mucus and in turn CFTR dysfunction. In addition to pharmaceutical interventions, patients with CFTR dysfunction, in particular CF patients, will typically undergo physiotherapy to the chest and/or abdomen designed to alleviate the lung and/or GI complications, particularly in relation to assisting the clearing of the lungs and/or breathing. Such physiotherapy techniques may include one or more of active cycle of breathing techniques (ACBT), postural drainage, manual percussion and vibration, autogenic drainage (AD), high frequency chest wall oscillation (HFCWO), positive expiratory pressure (PEP), and oscillating positive expiratory pressure devices (Oscillating PEP).
In patients with CFTR dysfunction, lung complications are typically managed through antibiotic, antifungal, antiinflammatory and bronchodilator treatment regimes, the nutrient malabsorption caused by pancreatic complications can be treated with digestive enzyme supplements and cystic fibrosis-related diabetes may be treated by a combination of oral antidiabetic drugs (e.g. the sulfonylureas, biguanides and thiazolidinediones) and i.v. insulin. Liver complications are typically tackled as for other patients with liver disease, but little can be done once damage has occurred to any of these organs.
A few approaches have been developed to address the abnormalities of the mucus, principally its elevated viscosity. These include dornase alfa (a DNase enzyme), hypertonic saline, mannitol, acetylcysteine, dextran and denufosol (an agonist of the P2Y2 subtype of purinergic receptors, an alternative chloride channel in the lung). However, these treatments only show limited efficacy and are limited to the lung. Alginate oligomers have also been shown to be capable of reducing the viscosity of sputum from COPD patients and cervical mucus (WO 2007/039754, WO 2007/039760; WO2008/125828) and the use of alginate oligomers to treat CF, female infertility and hyperviscous mucus in the gut has been proposed on this basis.
Alginates are naturally occurring polysaccharides that have been found to have a number of uses, both clinical (e.g. in wound dressings, as drug carriers and in anti-heartburn preparations) and non-clinical (e.g. in food preparation). They are linear polymers of (1-4) linked β-D-mannuronic acid (M) and/or its C-5 epimer α-L-guluronic acid (G). The primary structure of alginates can vary greatly. The M and G residues can be organised as homopolymeric blocks of contiguous M or G residues, as blocks of alternating M and G residues and single M or G residues can be found interspacing these block structures. An alginate molecule can comprise some or all of these structures and such structures might not be uniformly distributed throughout the polymer. In the extreme, there exists a homopolymer of guluronic acid (polyguluronate) or a homopolymer of mannuronic acid (polymannuronate).
Alginates have been isolated from marine brown algae (e.g. certain species of Durvillea, Lessonia and Laminaria) and bacteria such as Pseudomonas aeruginosa and Azotobacter vinelandii. Other pseudomonads (e.g. Pseudomonas fluorescens, Pseudomonas putida, and Pseudomonas mendocina) retain the genetic capacity to produce alginates but in the wild they do not produce detectable levels of alginate. By mutation these non-producing pseudomonads can be induced to produce stably large quantities of alginate.
Alginate is synthesised as polymannuronate and G residues are formed by the action of epimerases (specifically C-5 epimerases) on the M residues in the polymer. In the case of alginates extracted from algae, the G residues are predominantly organised as G blocks because the enzymes involved in alginate biosynthesis in algae preferentially introduce the G neighbouring another G, thus converting stretches of M residues into G-blocks. Elucidation of these biosynthetic systems has allowed the production of alginates with specific primary structures (WO 94/09124, Gimmestad, M et al, Journal of Bacteriology, 2003, Vol 185(12) 3515-3523 and WO 2004/011628).
Alginates are typically isolated from natural sources as large high molecular weight polymers (e.g. an average molecular weight in the range 300,000 to 500,000 Daltons). It is known, however, that such large alginate polymers may be degraded, or broken down, e.g. by chemical or enzymatic hydrolysis to produce alginate structures of lower molecular weight. Alginates that are used industrially typically have an average molecular weight in the range of 100,000 to 300,000 Daltons (such alginates are still considered to be large polymers) although alginates of an average molecular weight of approximately 35,000 Daltons have been used as excipients in pharmaceuticals.
In addition to a proposed use of alginate oligomers of smaller size (molecular mass) to reduce the viscosity of hyperviscous sputum such as occurs in sufferers of cystic fibrosis and other respiratory diseases (see WO 2007/039754 and WO 2008/125828), such oligomers have also been proposed for other clinical uses, to combat biofilm (WO 2009/068841) and multidrug resistant bacteria (WO 2010/13957).
More recently the new pharmaceutical class of “CFTR modulators” has emerged offering a pharmaceutical intervention at the level of CFTR dysfunction, in particular in the treatment of CF (Derichs, N., Eur. Respir. Rev., 2013, 22(127), 58-65; Petit, R. S. and Fellner, C., Pharmacy and Therapeutics, 2014, 39(7), 500-511). Also known as “CFTR modifiers”, which terms are used interchangeably herein, CFTR modulators are small molecules which can redress, at least partially, a mechanism of CFTR dysfunction from one or more classes of CFTR dysfunction. Present CFTR modulators fall into three main groups: CFTR potentiators, CFTR correctors and read-through agents.
CFTR potentiators are CFTR modulators which increase the activity of the CFTR ion channel present on the epithelial cell surface (e.g. by increasing the open probability or conductance of the channel) and thus have utility in contexts in which a Class III or a Class IV dysfunction is present (i.e. a dysfunction caused by gating or conductance problems in the CFTR at the cell surface). Prototypical examples of CFTR potentiators are ivacaftor (VX-770; N-(2,4-di-tert-butyl-5-hydroxyphenyl)-1,4-dihydro-4-oxoquinoline-3-carboxamide) and VRT-532 (4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol) of Vertex Pharmaceuticals).
CFTR correctors are CFTR modulators which increase the amount of CFTR protein delivered or retained at the epithelial cell surface. These molecules may achieve this effect in a variety of ways in view of the variety of defects in the processing of CFTR that can cause reduced quantities of CFTR at the epithelial cell surface. For instance, certain CFTR correctors can act as a chaperone facilitating proper folding and post-translational modification of CFTR, protecting CFTR from premature degradation, facilitating intracellular targeting of CFTR and reversing accelerated turnover of CFTR at the cell membrane. Correctors thus have utility in the context of Class II, Class V and Class VI dysfunctions. Prototypical examples of CFTR correctors include lumacaftor (VX-809) and tezacaftor (VX-661) of Vertex Pharmaceuticals and N6022 (3-[1-(4-carbamoyl-2-methylphenyl)-5-(4-imidazol-1-ylphenyl)pyrrol-2-yl]propanoic acid).
Read-through agents (also known as “premature stop codon suppressors” (PSC suppressors) or “premature termination codon suppressors” (PTC suppressors, which terms are used interchangeably herein) are CFTR modulators which cause the translation machinery of the cell to pass over any premature termination codon in the CFTR mRNA thereby increasing the amount of substantially full length and functional CFTR produced. Read-through agents thus have utility in the context of Class I dysfunctions. Prototypical examples of read-through agents include ataluren (PTC124) of PTC Therapeutics and gentamicin.
Further CFTR modulators are disclosed in WO2006/002421, WO2007/056341 WO2007134279, WO2009038683, WO2009064959, WO2009073757, WO2009076141, WO2009076142, WO2010019239, WO2010037066, WO2010048526, WO2010053471, WO2010054138, WO2010138484, WO2011019413, WO2011050325, WO2011072241, WO2011127241, WO2011127290, WO2011133751, WO2011133951, WO2011133953, WO2011133956, WO2011146901, Pedemonte, N., et al., J Clin Invest. 2005; 115(9):2564-2571, Van Goor, F. et al., Am J Physiol Lung Cell Mol Physiol 2006, 290: L1117-L1130, and Pedemonte, N., et al., Molecular Pharmacology, 2005 vol. 67 no. 5 1797-1807 the content of which is incorporated herein by reference, and FIG. 3.
Experience with CFTR modulators to date has been based on systemic administration via oral or injection routes, at least in part in order to avoid potential bioavailability complications arising from the possible “barrier effect” of the abnormal mucus which results from CFTR dysfunction, namely that the presence of the abnormal mucus may impede access of the modulator to the epithelial cells (especially in the respiratory system where bioburden is significantly greater than at other mucosal surfaces, but also in the GI tract where the mucus layer is comparatively thicker than at other mucosal surfaces). Moreover, it has been suggested that younger patients will respond better to this class of therapeutic agent because the younger a patient is, the less damage has accrued. More particularly, in younger patients there has been less time for a CFTR dysfunction phenotype (especially one including chronic infection, chronic inflammation and airway remodelling) to become established and to develop into a phenotype that can interfere with the action of CFTR modulators. Administration by inhalation, for instance, to a patient with a well-established CFTR dysfunction phenotype would, for example, be considered very challenging.
Although existing developments in this field show promise, there is a continuing need for improved pharmaceutical interventions, including treatment regimens, for conditions arising from or associated with CFTR dysfunction and, specifically, the abnormal mucus of patients with CFTR dysfunction (e.g. patients with CF, patients with abnormal mucus clearance in the respiratory tract and/or breathing difficulties resulting from chronic particulate inhalation, patients with chronic inflammatory respiratory disorders, e.g. COPD (and its subtypes chronic bronchitis and emphysema), bronchiectasis, asthma and chronic sinusitis, and/or patients with non-compound CFTR gene mutation) especially those conditions associated with the lung, the GI tract, the pancreas, the liver and the reproductive system.